PROJECT SUMMARY Natural killer (NK) cells are innate lymphocytes that make up 10-15% of the circulating lymphocytes in the blood. They are important in host defense against viruses and cancerous cells, and treatments that augment NK cell function in humans are currently in clinical trials. A better understanding of how NK cells are regulated will allow development of new strategies to manipulate NK cell function and improve NK cell-based therapies. Recent studies have demonstrated that metabolism regulates immune cell differentiation and function. Studies in T cells, macrophages, and dendritic cells have shown that activation induces metabolic reprogramming, in which cells change what metabolic pathways they use to fuel their activities. Reprogramming is required for these cells for specific functions, such as production of effector molecules or proliferation. In vitro studies using drug inhibitors and nutrient deprivation have suggested that NK cells are similarly reprogrammed under certain activating conditions, and that NK cells rely on glycolysis for multiple functions. The goal of this proposal is to test whether NK cells require high glycolytic flux to control viral infection in vivo. Preliminary studies indicate that specific NK cell responses to viral infection with murine cytomegalovirus (MCMV) are decreased by global glycolytic inhibition with a drug. However, new systems are required to investigate NK cell specific metabolic pathways in vivo. To examine the role of glycolysis during viral infection without the concern of off-target drug effects, this application proposes to use an NK-specific knockout of an enzyme that regulates glycolysis. Aim 1 will quantify the development and function of NK cells genetically deficient in glycolysis, as well as examine their reliance on alternate metabolic fuels and pathways at baseline and after stimulation. Aim 2 will characterize the glycolytic requirements for NK cell responses to MCMV in vivo. Susceptibility to infection will be examined, as well as NK cell cytokine production, proliferation, target cell lysis, and memory formation. These aims will characterize a new model for examining metabolic requirements of immune cells that is amenable to in vivo experiments, and elucidate the role that glycolysis plays in NK cell responses to viral infection. This work will contribute to a greater understanding of how metabolism and classical NK cell activating signals interact, as well as shed light on possible methods to boost or dampen NK cell function by altering their metabolism.